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Research ArticleCELLULAR AND MOLECULAR

Agonist-Directed Trafficking of Porcine α2A-Adrenergic Receptor Signaling in Chinese Hamster Ovary Cells: l-Isoproterenol Selectively Activates Gs

Christiaan B. Brink, Susan M. Wade and Richard R. Neubig
Journal of Pharmacology and Experimental Therapeutics August 2000, 294 (2) 539-547;

Abstract

In this study, we investigated the hypothesis of agonist-directed trafficking of receptor signaling for the α2A-adrenergic receptor (α2A-AR). α2A-ARs couple to both Gs and Gi to stimulate or inhibit adenylyl cyclase activity. Chinese hamster ovary-K1 cell lines expressing the porcine α2A-AR at high (α2A-H) and low (α2A-L) levels were used to estimate the relative efficacies (R.e.s) of a series of agonists for the Gs and Gi pathways. Gs-mediated responses were measured after pertussis toxin treatment to inactivate Gi in α2A-H, whereas Gi responses were measured in α2A-L, where Gs responses were absent. The full agonist UK-14,304 showed a large receptor reserve for Gi responses in α2A-H but little receptor reserve for Gs responses in α2A-H or for Gi responses in α2A-L. With the exception ofl-isoproterenol (ISO), all agonists showed similar R.e.s at the α2A-AR for Gs and Gi responses, with rank orders of R.e.s as follows: l-epinephrine =l-norepinephrine = UK-14,304 >p-aminoclonidine ≥ BHT-920 ≥ BHT-933 > clonidine = p-iodoclonidine ≥ xylazine ≥ guanabenz. Interestingly, ISO had the highest efficacy at the α2A-AR for activating Gs versus Gi (9-fold higher); however, it had low potency for both. By several criteria, the ISO response was mediated by the α2A-AR, supporting the hypothesis of agonist-directed trafficking of receptor signaling or agonist-specific G protein selectivity. In contrast, the apparent Gi pathway selectivity of oxymetazoline appears to be mediated by an endogenous serotonergic receptor. It is intriguing that a classic β-AR agonist that activates Gs through β2-ARs also appears to produce a Gs-selective conformation of the Gi-coupled α2A-AR.

The hypothesis of agonist-directed trafficking of receptor signaling (ADTRS; Kenakin, 1995) predicts that when a receptor signals through two or more independent signal transduction pathways, the relative efficacies (R.e.s) of a series of agonists may differ for the pathways. This contrasts with the classic concept that intrinsic efficacy is a solely agonist-dependent pharmacodynamic parameter (Furchgott, 1966). This hypothesis builds on the ideas that a receptor can exist in distinct states (or conformations) and that the ability of those states to activate different G protein types or subtypes may differ.

The idea of ADTRS led to the development of the “N-state receptor models,” in which the receptor is assumed to exist in N states that may be “inactive” (R) or “active” (R∗, R∗∗, etc.). As a special case of the N-state receptor models, Leff et al. (1997)developed a mathematical model for three receptor states that accommodates the concept of ADTRS. They were able to predict differential R.e.s from this model. In this regard, Berg et al. (1998) recently reported strong evidence for the existence of pathway-dependent R.e.s for a series of five agonists at 5-hydroxytryptamine (5-HT)2A and 5-HT2C receptors when looking at the phospholipase C and phospholipase A2 signal transduction pathways. They also pointed out that for ADTRS to be possible, it is critical that the independent pathways diverge at the receptor/G protein level (e.g., receptor activates two G proteins independently, leading to the transduction of the stimulus through two separate effector pathways) and not downstream from a common G protein (e.g., receptor activates a G protein such as Gq that subsequently activates phospholipase C, which in turn activates two independent effector pathways via release of inositol trisphosphate and diacylglycerol).

According to the three-state model by Leff et al. (1997), two cases can be distinguished. The first is when the two effector pathways result in two independently measurable responses, as in the work of Berg et al. (1998) mentioned earlier. Second, the two pathways can diverge and eventually recombine to modulate one measurable response (e.g., regulation of adenylyl cyclase by both Gs and Gi proteins). If one pathway is isolated by inactivating the other selectively, R.e.s can be determined for the active pathway. It should now be possible to obtain an estimate of the R.e.s for each pathway independently, and theoretically their R.e.s may be different.

α2A-Adrenergic receptors (α2A-ARs) have previously been shown to activate three G proteins: Gi, Gs, and Gq (Eason et al., 1992; Chabre et al., 1994). The former study reported a pertussis toxin-insensitive stimulation of cAMP accumulation in Chinese hamster ovary (CHO) cells expressing high amounts of α2-AR. In addition to functional studies, direct, agonist-dependent, physical coupling of the α2A-AR to Gs was demonstrated (Eason et al., 1992). For the Gqcoupling, Chabre et al. (1994) used human embryonic kidney (HEK) 293 cells transiently transfected with the porcine α2A-AR and murine Gαq or rat Gαs. They estimated that the efficiency of coupling of the α2A-AR to endogenous Gi was approximately 1000 times higher than that to Gs or Gq.

Although no conclusive evidence was provided to support the hypothesis that ADTRS may occur for α2-ARs, three previous reports suggested such a possibility. Using CHO cells expressing the human α2A-AR (α2C10),Eason et al. (1994) showed a rank order of intrinsic activities of agonists for the Gs signal transduction pathway to be epinephrine = norepinephrine = UK-14,304 > BHT-933 > BHT-920 > oxymetazoline. For the Gi signal transduction pathway, they found that the intrinsic activities of these agonists were remarkably similar. They did not, however, take into account receptor reserve, so the true relative efficacies of these drugs cannot be determined from their data. Kenakin (1995) found that oxymetazoline selectively activated Gi (compared with Gs) in CHO cells stably transfected with α2A-AR. Yang and Lanier (1999) found differential regulation of Go and Gi by clonidine and epinephrine. A potential mechanism for ADTRS has been suggested by reports that different amino acid residues of the α2A-AR are required for Gi and Gs activation (Eason and Liggett, 1996; Wade et al., 1999).

In the current study, we asked whether convincing evidence for the ADTRS hypothesis could be found for the regulation of Gi and Gs by the α2A-AR. To account for the difference in spare receptors (for a full agonist) when measuring Gsor Gi responses, we used two CHO-K1 cell lines: one expressing the porcine α2A-AR at high levels and the other expressing the porcine α2A-AR at low levels. We estimated the R.e.s for a series of agonists for the Gs and Gi signal transduction pathways, measuring [3H]cAMP accumulation as the response. Interestingly, l-isoproterenol (ISO), but not oxymetazoline, exhibits ADTRS.

Experimental Procedures

Materials

Radiochemicals.

[2-3H]Adenine (21–25 Ci/mmol) was obtained from Amersham Life Science (Piscataway, NJ). [3H]Yohimbine (74.5–78 Ci/mmol) was obtained from DuPont-New England Nuclear (Boston, MA).

Other Chemicals.

cAMP, ATP, 3-isobutyl-1-methyl-xanthine (IBMX), l-epinephrine-(+)-bitartrate, ISO, and yohimbine HCl were purchased from Sigma (St. Louis, MO). UK-14,304,p-iodoclonidine HCl, l-norepinephrine bitartrate, BHT-933 dihydrochloride, xylazine HCl, and (S)-(−)-cyanopindolol hemifumarate were obtained from Research Biochemicals Inc. (Natick, MA). Clonidine HCl andp-aminoclonidine were purchased from Boehringer Ingelheim (Ingelheim, Germany). Guanabenz acetate was obtained from Wyeth Laboratories (Philadelphia, PA). BHT-920 Cl2 was purchased from Dr. Karl Thaomae Inc. (Biberach, Germany). Oxymetazoline HCl was purchased from Schering Corporation (Bloomfield, NJ). Propranolol HCl was purchased from Ayerst Laboratories Inc. (New York, NY). Benextramine tetrahydrochloride monohydrate was obtained from Aldrich (Milwaukee, WI). Pertussis toxin (PTX) was obtained from List Biological Laboratories (Campbell, CA). Forskolin was obtained from Calbiochem-Novabiochem Corp. (San Diego, CA). LipoFECTAMINE and geneticin (G418) were obtained from Life Technologies (Gaithersburg, MD). Fluorescein-conjugated 12CA5 anti-hemagglutinin monoclonal antibody was purchased from Boehringer Mannheim (Indianapolis, IN).

α2A-AR Expressing Cell Lines

In this study, we used several cell lines. The first are CHO-K1 cell lines expressing high concentrations of either the wild type or one of two mutant porcine α2A-ARs (Wade et al., 1999). All receptor constructs contain an amino-terminal HA tag. The receptor concentrations were estimated as 19 ± 2 pmol/mg membrane protein for the wild-type α2A-AR (clone 1 designated α2A-H in this report), 10 ± 1 pmol/mg for the R3 mutant α2A-AR (mutating RWRGR to AWAGA at residues 361–365 of the receptor, designated α2A-R3 in this report), and 36 ± 3 pmol/mg for the B2 mutant (mutating basic residues 368–371 of the membrane-proximal i3c region to form BXAA, designated α2A-B2 in this report). The α2A-R3 and α2A-B2 mutations disrupt coupling to Gs and GI, respectively (Wade et al., 1999).

For the purpose of this study, we isolated two additional CHO-K1 cell lines expressing a low concentration of the wild-type porcine α2A-AR. This was done with another flow cytometry sorting selection from the original transformation of Wade et al. (1999) from which the WT (clone 1) had been isolated. The cell line expressing the lowest concentration of the α2A-AR, as determined by [3H]yohimbine binding (Bmax ∼1 pmol/mg, clone 101), was selected for this study (designated α2A-L). This line exhibited a similar EC50 value for the dose-response curve of UK-14,304 through the Gipathway as did α2A-H through the Gs pathway, simplifying comparison of the Gs and Gi responses. The Neo cells containing the selection plasmid but no α2A-AR vector were used as controls.

Measurement of Whole-Cell [3H]cAMP Accumulation

CHO-K1 cells were maintained in Ham's F-12 medium with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2. Selection for stable expression was maintained by the addition of 0.4 mg/ml G418 (active). [3H]cAMP accumulation was determined in whole cells in 24-well plates as described by Wade et al. (1999), adding 1 μCi/well [3H]adenine and, when indicated, 100 ng/ml PTX for at least 18 h before the assay. Cells were then washed once with Dulbecco's modified Eagle's medium (DMEM), after which the assay was initiated by adding DMEM with 1 mM IBMX and 30 μM forskolin and the appropriate drug or drugs. After a 20-min incubation, the medium was aspirated, and the reaction was terminated with 1 ml of 5% trichloroacetic acid (TCA) containing 1 mM ATP and 1 mM cAMP. The acid-soluble nucleotides were separated on Dowex and alumina columns as described by Salomon et al. (1974). The cAMP accumulation was normalized by dividing the [3H]cAMP counts by the total [3H]nucleotide counts. The control percent conversion of ATP to cAMP was 10 to 14% for non-PTX-treated cells (n = 81) and 3.7 to 7.4% for PTX-treated cells (n = 66) and did not vary significantly for high, low, or no α2A-AR expression. This percent conversion value was then divided by the corresponding value obtained with only IBMX and forskolin and no drug (to calculate percent of control).

Ligand Binding Assays

The Ki values of the α2A-AR agonists were determined from competition binding curves in whole cells against 5 nM [3H]yohimbine (KD = 3 nM). The buffer used in these binding studies was comparable with that used for measuring cAMP accumulation. Cells were plated and incubated as before but without [3H]adenine and PTX. Cells were then washed once with OptiMEM, after which the assay was initiated by adding OptiMEM with 5 nM [3H]yohimbine and different concentrations of the testing drug. After a 30-min incubation, the medium was aspirated, the cells were washed twice, and the reaction was terminated with 1 ml of 5% TCA and allowed to stand for at least 30 min to allow the cells to lyse. The TCA from each well was then transferred directly into scintillation vials using transfer pipettes, and the [3H]yohimbine was counted.

Pharmacological Receptor Inactivation

Benextramine was used as an irreversible competitive antagonist of α2A-ARs. The cells were incubated overnight in 24-well plates as described earlier. The cells were then washed once with DMEM and incubated with 0, 1, 10, or 100 μM benextramine in PBS (containing 0.8% NaCl, 0.02% KCl, 0.09% Na2HPO4, and 0.02% KH2PO4) for 20 min at room temperature. This was followed by two washes with DMEM. With the second wash, the cells were let to stand in the DMEM for at least 5 min to ensure that all unbound benextramine was removed. The measurement of [3H]cAMP accumulation then proceeded as described earlier.

Data Analysis

Functional data, except where indicated otherwise, were obtained from three or more independent and comparable experiments, each in triplicate, and expressed as mean ± S.E.

Analyses of dose-response curves were made with the nonlinear least-squares method of the computer program Prism (GraphPad Software, San Diego, CA), setting the Hill slope factor at 1. Results are expressed as the mean ± S.E. To verify statistical significance of differences between mean values, the nonparametric Student'st test was used. After the Bonferroni correction for multiple comparison, a value of P < .05 was taken as statistically significant.

The relative intrinsic activity (RIA) was determined from functional data and expressed as the maximal response (Emax) of an agonist relative to that of the full agonist UK-14,304. The apparent dissociation constant of an agonist-receptor complex [KA(app.)], and the fraction of receptors still functional after partial receptor alkylation (q) was estimated from Furchgott analysis of dose-response curves of UK-14,304 before and after partial receptor alkylation (Furchgott, 1966).

Because there appeared to be a small receptor reserve remaining for the full agonists (i.e., l-epinephrine,l-norepinephrine, UK-14,304) in α2a-L for Gi and α2a-H for Gs responses, we estimated the R.e.s of full agonists as follows:R.e.=E50(drug)/E50(UK­14,304) where E50 is the response at a drug concentration equal to its Ki value. Relative efficacies of partial agonists were determined as:R.e.=Emax(drug)/Smax(UK­14,304) where Smax andEmax are as defined later.

This analysis for full agonists is similar to that used by Van Rossum (1966) and Van den Brink (1977) to define a “corrected” intrinsic activity constant αS or by Venter (1997) to define the efficacy-related parameter eES. It assumes that 50% of the functionally coupled receptors are occupied at a concentration of agonist equal to theKi value and that the relationship between receptor occupation and stimulus is linear over the range studied. Because the maximal stimulus should be proportional to twice the stimulus at the Ki, we also defineSmax to equal two times the response (linearly related to stimulus) obtained at the E50. Supporting this assumption of linearity, theSmax/Emaxvalue for UK-14,304 determined in this manner is also similar to theSmax/Emaxvalue from the q value estimated from Furchgott analysis before and after partial receptor alkylation (see above), whereEmax was obtained before partial receptor alkylation. In this case,Smax was estimated fromEmax′/q, whereEmax′ represents the reducedEmax after partial receptor alkylation.

Results

α2A-ARs Expressed in α2A-H and α2A-L Are Comparable Pharmacologically.

From saturation binding with [3H]yohimbine, theBmax values in α2A-H and α2A-L cells were 2.0 ± 0.1 × 106 and 0.27 ± 0.09 × 106 α2A-ARs per cell, respectively. Thus, α2A-H cells express ∼10 times more of the porcine α2A-AR than α2A-L cells. The pKD values for yohimbine in α2A-H and α2A-L were similar, being 8.57 ± 0.06 and 8.57 ± 0.24, respectively.

In the absence of PTX, Gi-mediated inhibition of adenylyl cyclase by the full agonist UK-14,304 predominates over the Gs response (Fig.1, bottom). The EC50 value for UK-14,304 in the α2A-H cells (−PTX) is ∼1000-fold lower than the Ki value determined from [3H]yohimbine competition (Table1), suggesting that there is a large receptor reserve. The EC50 value of the dose-response curve for α2A-L (−PTX) is 2 logs higher than that in α2A-H, which is expected with the lower receptor expression and therefore smaller receptor reserve for the α2A-L.

Figure 1
Figure 1

The α2A-AR couples to Gi and Gs in α2A-H and α2A-L. Whole-cell [3H]cAMP accumulation measurements with increasing concentrations of UK-14,304 were made for a high-expressing cell line, α2A-H (▴, ●), or for a lower-expressing cell line, α2A-L (○). Cells were pretreated overnight with (top) or without (bottom) 100 ng/ml PTX. The data are averages of triplicate measurements from at least three experiments in each cell line. They are expressed as percent of control samples with no agonist.

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Table 1

The KA(app.) and q values for UK-14,304 as calculated from Furchgott analysis of dose-response curves of UK-14,304 in α2A-H (+ PTX ) and α2A-L (− PTX ) before and after partial receptor alkylation by the irreversible inhibitor benextramine

In α2A-H (−PTX), at concentrations of UK-14,304 of greater that 10−8 M, a modest stimulation of adenylyl cyclase is seen, confirming the Gs protein activation previously reported (Eason et al., 1992; Wade et al., 1999). In α2A-L (−PTX), the stimulation of adenylyl cyclase is not observed. After PTX treatment, which inhibits receptor activation of Gi, only the Gs-mediated stimulation of adenylyl cyclase can be seen in α2A-H (Fig. 1, top). However, even in the presence of PTX, the stimulation of adenylyl cyclase is not evident in α2A-L (+PTX). Thus, the inhibition of adenylyl cyclase in α2A-L (−PTX) will not be functionally antagonized by Gs-mediated stimulation, as may be occurring in α2A-H (−PTX). The 120-fold difference in the EC50values for the dose-response curves of UK-14,304 for the Gs versus the Gi pathways [i.e., in α2A-H (+PTX) versus α2A-H (−PTX), respectively] suggests a much more efficient coupling of the α2A-AR to Gi than to Gs. It should also be noted that stimulation of adenylyl cyclase in α2A-H (+PTX) occurs at nearly the same concentration of UK-14,304 as does inhibition in α2A-L (−PTX). Although not a prerequisite for comparison, this renders these two cell lines ideal for comparison of Gs- and Gi-mediated responses.

We investigated the receptor reserve for UK-14,304 in α2A-H (±PTX) and α2A-L (−PTX) by irreversible inhibition of α2A-ARs by benextramine. The EC50 value for the dose-response curves in α2A-H (−PTX) before benextramine treatment is more than 1000-fold lower than theKi value for UK-14,304 binding in cells, and the curves are shifted progressively to the right in a parallel fashion by increasing concentrations of benextramine (Fig.2B). Even 100 μM benextramine for 20 min is not sufficient to eliminate receptor reserve as theEmax is maintained and the EC50 is still ∼10-fold lower than theKi value. The EC50 values for the dose-response curves in α2A-H (+PTX) and α2A-L (−PTX) before benextramine treatment are ∼6- and ∼4-fold lower, respectively, than the Ki value (Fig.2, A and C). With 1 μM benextramine treatment, these curves are shifted to the right with EC50 values comparable with the Ki value. TheEmax is also decreased, indicating that the receptor concentration is decreased sufficiently to eliminate receptor reserve with even the smallest concentration of benextramine. Results from Furchgott analysis of the data in α2A-H (+PTX) and α2A-L (−PTX) are presented in Table 1.

Figure 2
Figure 2

Effect of the irreversible α2A-AR inhibitor benextramine on responses in α2A-H and α2A-L cell lines. Whole-cell [3H]cAMP accumulation measurements were made with increasing concentrations of the agonist UK-14,304 after a 20-min treatment with 0 (●), 1 μM (▵), 10 μM (○), or 100 μM (⋄) of the irreversible inhibitor benextramine. Cells used were α2A-H (+PTX) (A), α2A-H (−PTX) (B), and α2A-L (−PTX) (C). Data are averages of triplicate measurements from four experiments in α2A-H (+PTX), one experiment in α2A-H (−PTX), and two experiments in α2A-L (−PTX). Curves are nonlinear least-squares fits.

Binding Data for Selected α2A-AR Agonists.

According to classic theory, ∼95% receptor occupation, and thereforeEmax, is expected at a concentration of 20× Ki of an agonist. Thus, RIAs can be estimated from these single maximal concentrations of a series of agonists. Therefore, we calculated theKi values for a series of α2A-AR agonists from competition binding against 5 nM [3H]yohimbine (Table2). All competition binding curves, except for l-epinephrine, l-norepinephrine, and clonidine, were monophasic and could be explained by a single binding site. The competition binding curves for l-epinephrine,l-norepinephrine, and clonidine were biphasic with high-affinity pKi values being 6.19, 5.25, and 9.18, respectively (and 57, 41, and 23% of the [3H]yohimbine displaced, respectively) and low-affinity pKi values being 4.07, 3.58, and 7.07, respectively. For l-epinephrine andl-norepinephrine, the biphasic competition binding curves can be explained by their hydrophilic properties, presumably hindering access to nonsurface α2A-ARs. The high-affinity pKi would therefore be expected to describe binding to the surface receptors. After 1 or 10 μM benextramine treatment, the pEC50 values of the dose-response curves of l-epinephrine (data not shown) corresponded with the high-affinity pKi value as expected. Also, the high-affinity pKi values forl-epinephrine and l-norepinephrine were closest to the pEC50 values measured from functional dose-response curves in α2A-H (+PTX) and α2A-L (−PTX) (see later). However, for the partial agonist clonidine, the pEC50 values from functional dose-response curves (data not shown) corresponded best with the low-affinity pKi. We cannot explain the small fraction of high-affinity clonidine binding sites observed here.

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Table 2

The Ki values, pEC50 values, R.I.A., and R.e. of selected agonists on α2A-ARs for the Gs and Gi pathways

Comparison of Gs- and Gi-Mediated Signaling.

α2A-H (+PTX) and α2A-L (−PTX) were used to estimate RIAs of a series of full and partial agonists for the Gs- and Gi-mediated modulation of adenylyl cyclase activity. Because the α2A-AR couples more effectively to Gi than to Gs, [3H]cAMP production was measured in the lower receptor expressing α2A-L cells to examine the Gi pathway and in the higher receptor expressing α2A-H cells treated with PTX (+PTX) for the Gs pathway. Neo cells without the porcine α2A-AR were used as controls.

Dose-response curves in α2A-H (+PTX) and α2A-L (−PTX) are presented in Fig.3. Of the agonists studied,l-epinephrine, l-norepinephrine, and UK-14,304 behaved as full agonists and BHT-920 behaved as a partial agonist for both the Gs- and Gi-mediated responses. These agonists showed no significant differences in the relative maximum responses between the Gi and Gs pathways. However, oxymetazoline showed selectivity for inducing signaling through the Gi pathway compared with the other partial agonist BHT-920. Very interestingly, ISO showed clear selectivity for inducing signaling through the Gspathway.

Figure 3
Figure 3

Dose-response curves of various agonists for the Gs and Gi pathways. Whole-cell [3H]cAMP accumulation measurements with increasing concentrations of the agonists l-epinephrine (●),l-norepinephrine (○), UK-14,304 (♦), BHT-920 (⋄), oxymetazoline (∗), and l-isoproterenol (▴) were performed on α2A-H cells (+PTX) and α2A-L cells (−PTX). A, C, E, and G, Gs responses (α2A-H, +PTX). B, D, F, and H, Gi responses (α2A-L, −PTX). The EC50 values are reported in Table 2. Gs- and Gi-mediated responses show comparable relative intrinsic activities with the exception ofl-isoproterenol and oxymetazoline. Data are averages of triplicate measurements from three experiments.

Having the two agonists, oxymetazoline and ISO, show opposite selectivity for the Gi and Gs pathways, it was important to investigate whether these responses were mediated via interaction with α2A-ARs. For this purpose, we used the control Neo cells without the porcine α2A-AR to study dose-response curves for oxymetazoline and ISO. It can be seen in Fig.4B that the inhibition of adenylyl cyclase by oxymetazoline also occurs in Neo cells, suggesting that this response is mediated by a receptor type other than the α2A-AR. Because the inhibition of adenylyl cyclase is not seen after PTX treatment of Neo cells, this receptor is signaling through a PTX-sensitive G protein (e.g., Gi). The Gs-mediated stimulation of adenylyl cyclase by oxymetazoline, however, is mediated by α2A-ARs, because no response is seen in Neo cells (Fig. 4A). It is clear from Fig. 4C that ISO is inducing its Gs selective response via α2A-ARs because no response is seen in Neo cells. The Gi response to ISO is small but also appears to be mediated by the α2A-AR.

Figure 4
Figure 4

The Gs response ofl-isoproterenol, but not the Gi response of oxymetazoline, is mediated by α2A-ARs. Whole-cell [3H]cAMP accumulation measurements with increasing concentrations of (A and B) oxymetazoline and (C and D)l-isoproterenol were performed on α2A-H (+PTX) and α2A-L (−PTX) (●, ♦) and compared with the corresponding responses in Neo (±PTX) (○, ⋄). The Gs-mediated stimulation of adenylyl cyclase byl-isoproterenol in α2A-H (+PTX) is mediated by α2A-ARs, because Neo (+PTX) shows no response. However, enhanced oxymetazoline inhibition of adenylyl cyclase is mediated by an endogenous Gi-coupled, non-α2-AR (see Neo cells). Data are averages of triplicate measurements from three experiments.

The list of agonists to be evaluated for Gs- and Gi-mediated responses in α2A-H (+PTX) and α2A-L (−PTX) was also extended. By assuming that anEmax is obtained at a concentration of agonist 20 times the Ki value as also used by Berg et al. (1998), RIAs of the agonists were determined for the Gs and Gi pathways and are reported in Table 2. The RIAs of the full agonists were similar, and the rank order of RIA for the Gs pathway can be given as l-epinephrine =l-norepinephrine = ISO = UK-14,304 >p-aminoclonidine > BHT-920 = BHT-933 > clonidine > xylazine = guanabenz > oxymetazoline. The small differences in the Emax values for the full agonists were all statistically insignificant after the Bonferroni correction for multiple comparison. However, for the Gi pathway, the difference between theEmax values forl-epinephrine and UK-14,304, but not forl-norepinephrine and UK-14,304, are statistically significant (P < .01 after the Bonferroni correction for multiple comparison). Some of these differences may be due to a small stimulation of adenylyl cyclase in the Neo cells (Table3). l-Epinephrine,l-norepinephrine, p-iodoclonidine, and oxymetazoline exhibited responses that were significantly greater than zero (P < .05 after Bonferroni's correction for multiple comparison) in Neo (±PTX). Because the endogenous full agonists l-epinephrine and l-norepinephrine showed modest stimulation of adenylyl cyclase in both PTX-treated and untreated Neo cells, the RIAs of all agonists were calculated relative to UK-14,304.

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Table 3

The relative Emax(R.Emax) obtained at 20× Kiconcentrations of agonists in Neo cells (± PTX )

ISO Responses Are Blocked by α2A Antagonist Yohimbine but Not by β-Blocker Propranolol.

To further verify that the Gs response seen with ISO in α2A-H (+PTX) is mediated by interaction with α2A-ARs, we obtained data for dose-response curves for ISO in the absence and presence of the β-AR antagonist propranolol (because ISO is a classic β-AR agonist) or the α2-AR antagonist yohimbine. From Fig.5A, it can be seen that 1 μM propranolol does not inhibit the response by ISO, whereas 1 μM yohimbine greatly inhibits this response. This would exclude the contribution of any endogenous β-ARs to the observed stimulation of adenylyl cyclase by ISO. It also confirms that the Gs response is mediated by α2A-ARs, in agreement with data from the Neo cells. Results for the Gi response suggest the same with regard to the interaction of ISO with α2A-ARs (Fig. 5B).

Figure 5
Figure 5

Yohimbine, but not propranolol, blocks the response of l-isoproterenol. Whole-cell [3H]cAMP accumulation measurements with increasing concentrations ofl-isoproterenol for the Gs(α2A-H, +PTX) (A) and Gi(α2A-L, −PTX) (B) pathways without (▪) and with 1 μM β-AR antagonist propranolol (○) or 1 μM α2-AR antagonist yohimbine (▵). The data are averages of triplicate measurements from three experiments and indicate that the Gs-mediated response is mediated via α2A-ARs and not by β-ARs.

Oxymetazoline but Not UK-14,304 Responses Are Blocked by 5-HT1 Antagonist (−)-Cyanopindolol.

Figure6 shows dose-response curves of UK-14,304 and oxymetazoline in the presence and absence of 100 nM (−)-cyanopindolol in α2A-L (−PTX). The Gi response of oxymetazoline, but not that of UK-14,304, was antagonized by 100 nM (−)-cyanopindolol. The estimated pA2 value is 8.44, indicating a KB value of ∼3.6 nM for (−)-cyanopindolol for the receptor involved. These data suggest that 5-HT1 receptors may mediate much of the Gi response of oxymetazoline (see Discussion). Also, 100 nM propranolol did not shift the response of oxymetazoline to the right, excluding any contribution of β-ARs to the response. Thus, the apparent ADTRS by oxymetazoline is an artifact of endogenous receptors, whereas that of ISO reflects a true property of the α2A-AR.

Figure 6
Figure 6

Adenylyl cyclase inhibition by oxymetazoline, but not that by UK-14,304, is antagonized by (−)-cyanopindolol. Whole-cell [3H]cAMP accumulation measurements with increasing concentrations of UK-14,304 (A) in the absence (●) or presence of 100 nM (−)-cyanopindolol (○) and oxymetazoline (B) in the absence (●) or presence of 100 nM (−)-cyanopindolol (○) or 100 nM propranolol (∗). The data are averages of triplicate measurements from three experiments and indicate that a non-α2A-AR mediates the Gi-response of oxymetazoline.

Estimating Relative Efficacies of Agonists.

Because EC50/Ki ratios for l-epinephrine, l-norepinephrine, ISO, and UK-14,304 in α2A-H (+PTX) and forl-epinephrine, l-norepinephrine, and UK-14,304 in α2A-L (−PTX) suggest a small degree of receptor reserve for these full agonists, R.e. values cannot be estimated directly from Emaxvalues. This was further confirmed from dose-response curves of UK-14,304 before and after benextramine treatment (Fig. 2). Another complicating factor was the significant stimulation of adenylyl cyclase by l-epinephrine and l-norepinephrine seen in control Neo (±PTX) cells at concentrations of 20 times theKi value (Table 3).

At concentrations equal to their Kivalues, neither l-epinephrine norl-norepinephrine showed any response in Neo (±PTX) (data not shown), making this concentration suitable for measuring α2A-AR-mediated responses. Therefore, an alternative for measuring R.e.s would be to use submaximal responses at concentrations of the agonists equal to theirKi values (50% receptor occupation) as a measure of half-maximal stimulus and to calculate relativeSmax values from twice this response (see Experimental Procedures, Data Analysis).

One possible problem with this approach to keep in mind is the relationship between receptor occupation and response. This matter was clarified by estimating the KA(app.) values (see Table 1) from Furchgott analysis of the dose-response curves of UK-14,304 before and after benextramine treatment, as in Fig.2. At 1 and 10 μM concentrations of benextramine, theKA(app.) values were almost identical with the Ki values [seeKA(app.)/Kiratios in Table 1]. This would suggest a linear correlation between stimulus and response at concentrations of UK-14,304 yielding a submaximal response. Also, when the relativeSmax values were calculated from the fraction of receptors functional after alkylation (q) by 1 μM benextramine, it correlated very well with the relativeSmax values calculated from 1×Ki concentrations for both the Gs- and Gi-mediated responses. These results suggest that responses at 1×Ki concentrations can be used to estimate half-maximal stimulus (and therefore R.e.) in these cell lines. It should be noted that Furchgott analysis of dose-response curves of UK-14,304 before and after treatment with 100 μM benextramine gave KA(app.) values higher than Ki concentrations and the estimated relative Smax values differed greatly from those calculated from responses at a 1×Ki concentration of UK-14,304. Increasing concentrations of benextramine treatment not only decreased the Emax but also shifted the curves to the right beyond the Ki value, suggesting that the mechanism of inhibition at higher concentrations of benextramine may not be purely due to irreversible competitive antagonism. More reliable KA(app.) values are therefore calculated with the lowest concentration of benextramine (1 μM) where the least nonspecific effects are expected.

Although the responses for all full agonists at concentrations equal to their Ki values were submaximal, the responses were greater than 80% of theEmax values, and theoretically one may expect the relationship between stimulus and response to become nonlinear when receptor reserve is present. This would mean that the relative Smax values calculated from 1× Ki concentrations should be regarded as estimates.

The R.e.s of full agonists (relative to UK-14,304) were then calculated from Smax values equal to twice the response obtained at 1× Kiconcentrations of the agonists. The R.e.s of the partial agonists were calculated from the Emaxvalues obtained at 20× Kiconcentrations of the agonists. The R.e.s are reported in Table 2. From the R.e.s it can be seen that ISO shows a ∼9-fold higher efficacy for the Gs-mediated stimulation of adenylyl cyclase compared with the Gi-mediated inhibition of adenylyl cyclase.

α2A-AR Effector Site for Gs Coupling as Activated by ISO May Be Similar to that for Classic α2A-AR Agonists.

Because ISO is not a classic α2A-AR agonist and was the only agonist to show selectivity for activating the Gs pathway, the question arose of whether ISO induced an atypical G protein contact surface (or effector site) in the receptor for Gscoupling. Wade et al. (1999) recently showed that distinct α2A-AR regions are required for Gi and Gs activation. Using these mutant α2A-ARs that disrupt either Gs coupling (α2A-R3) or Gi coupling (α2A-B2), we investigated whether these mutations to the α2A-AR would disrupt the coupling to Gs and Gi in a similar fashion when ISO was used as agonist.

Figure 7 shows dose-response curves of UK-14,304 and ISO for both the Gs and Gi pathways in α2A-H (±PTX), α2A-R3 (±PTX), and α2A-B2 (±PTX). It is important to note that to permit comparison with mutant cell lines, the high expressing α2A-H line was needed for both Gi and Gs responses. Because of the high α2A-AR expression level in α2A-H, ISO behaves as a full agonist for the Gi response versus the partial agonism seen in the α2A-L cell line.

Figure 7
Figure 7

UK-14,304 and l-isoproterenol use similar effector regions on the α2A-AR for activation of Gs. Whole-cell [3H]cAMP accumulation measurements were made with increasing concentrations of UK-14,304 andl-isoproterenol. Shown are Gs (A and C, +PTX) and Gi (B and D, −PTX) responses with wild-type α2A-AR, ●, the α2A-R3 mutant ▵, or the α2A-B2 mutant ○ (see text for full explanation). Note that l-isoproterenol acts as a full agonist in the high α2A-AR expressing α2A-H (−PTX) cell line compared with being a partial agonist in α2A-L (−PTX) in other figures. Data are averages of at least triplicate measurements from three experiments.

The Gs responses of UK-14,304 and ISO were disrupted similarly by the α2A-R3 mutation. Likewise, the Gi responses of UK-14,304 and ISO were disrupted similarly by the α2A-B2 mutation. These data suggest that ISO activates a similar effector region of the α2A-AR for Gs coupling, as does the classic α2A-AR agonist UK-14,304. The effector region of the α2A-AR for Gicoupling also seems to be similar.

Discussion

The present study was undertaken to investigate agonist-directed trafficking of porcine α2A-AR signaling through the Gs- and Gi-coupled signal transduction pathways.

ISO Selectively Activates Gs Pathway by Interaction with α2A-ARs.

ISO was the only agonist tested to show clear selectivity for Gs or Gi at the α2A-AR. Interestingly, ISO had a 9-fold higher R.e. at the α2A-AR for Gs versus Gi, but it has low potency for both. It is clear from the data with both Neo control cells and pharmacological antagonists that this effect is mediated by the α2A-AR. These results support the hypothesis of ADTRS (Kenakin, 1995). We were not able to confirm the conclusion ofEason and Liggett (1996) that BHT-920 and BHT-933 lead to selective activation of Gi. The difference between these two conclusions is probably due to the fact that our analysis takes into account spare receptors, whereas theirs did not. A further difference is their use of human α2A-AR, whereas we have porcine receptors, although this does not seem to be a likely explanation. Other recently published reports of ADTRS include the study of Berg et al. (1998) in which 5-HT receptors couple to phospholipases C and A2 with efficacies dependent on the specific agonist; Bonhaus et al. (1998) found differential Gi and Gs coupling with cannabinoid (CB1) agonists, and Yang and Lanier (1999) found differential coupling of rat α2A-ARs to Go and Gi in NIH 3T3 cells.

It is intriguing that a classic β-AR agonist that activates Gs through β2-ARs also appears to produce a Gs-selective conformation of the α2A-AR, which typically activates Gi. We could not, however, demonstrate significant Gi- or Gs-mediated responses with other β2-AR agonists such as salbutamol orl-norephedrine because their affinities were too low (data not shown).

As a first step to understanding the possible conformational differences, we tested two recently described mutant α2-ARs that selectively alter Gi and Gs coupling. However, the Gs response in α2A-R3 and the Giresponse in α2A-B2 were inhibited similarly for UK-14,304 and ISO (see Fig. 7). We propose that the α2A-AR effector sites for Gs and Gi coupling when the α2A-AR is activated by ISO may be similar to those exposed when the α2A-AR binds other α2A-AR agonists.

Oxymetazoline Activates Gi Pathway by Interaction with Non-α2A-ARs.

Enhanced inhibition of adenylyl cyclase by oxymetazoline in CHO-K1 cells as previously reported by Kenakin (1995), appears to be mediated via an endogenous 5-HT receptor (Figs. 4and 6). Schoeffter and Hoyer (1991) estimated the affinity of oxymetazoline at 5-HT1B (rat cortex) receptors as a KD value of 26 nM. They also reported that stimulation of 5-HT1A, 5-HT1B, and 5-HT1Dreceptors inhibited adenylyl cyclase, whereas activation of 5-HT1C receptors stimulated adenylyl cyclase activity. Because CHO cells have an endogenous 5-HT1B receptor (Berg et al., 1994; Giles et al., 1996), that is the likely source of the excess Giresponse to oxymetazoline.

This was confirmed by the effects of cyanopindolol, a 5-HT1A/1B antagonist. (±)-Cyanopindolol binds the 5-HT1B receptor in rat brain membranes with aKi value of 3.5 ± 0.4 nM (Ariani et al., 1989). The pA2 of 7.76 (Fig.6) indicates a KB value of ∼18 nM for (−)-cyanopindolol for the receptor mediating this effect. This result strongly emphasizes the importance of nontransfected control cells when studying the pharmacological properties of cloned receptors.

Rank Order of Relative Efficacies for Gs and Gi Is Generally Similar.

With the exception of ISO, all agonists showed very similar R.e.s at the α2A-AR for the Gs and Gi pathways. There may be modest differences in R.e.s, such as BHT-920 for the Gi and Gs pathways (0.27 ± 0.02 and 0.19 ± 0.02, respectively), but this study did not have sufficient statistical power to define such a small difference. The rank order of R.e.s at the α2A-AR for activation of Gs and Gi pathways are as follows.

For the Gs pathway (rank order of R.e.s of oxymetazoline was not determined because of significant response in control Neo cells), the order isl-epinephrine = l-norepinephrine = ISO = UK-14,304 > p-aminoclonidine ≥ BHT-920 ≥ BHT-933 > clonidine =p-iodoclonidine ≥ xylazine ≥ guanabenz.

For the Gi pathway (rank order of R.e.s of p-iodoclonidine and oxymetazoline was not determined because of significant responses in control Neo cells), the order is l-epinephrine =l-norepinephrine = UK-14,304 >p-aminoclonidine ≥ BHT-920 > BHT-933 = xylazine ≥ clonidine ≥ ISO ≥ guanabenz.

The rank order of the R.e.s differs slightly from that found by Wise et al. (1997). They found that the R.e.s ofl-epinephrine and l-norepinephrine were greater than that of UK-14,304 [i.e., epinephrine (adrenaline) = norepinephrine (noradrenaline) = α-methylnoradrenaline > UK-14,304 > BHT-933 ≥ xylazine = clonidine]. However, their data were obtained in COS-7 cells transiently transfected to express the α2A-AR/Gi1αfusion protein, measuring GTPase activity as a readout of receptor activation. In CHO cells, Gαi2 and Gαi3 have been shown to mediate inhibition of adenylyl cyclase by the α2A-AR (Gerhardt and Neubig, 1991). It is possible that the difference in the Gα (i.e., Gi1 versus Gi2 and Gi3) may contribute to the differences seen in the R.e. of UK-14,304 and the catecholamines.

Defining α2A-H and α2A-L Pharmacologically.

The 120-fold difference in the EC50 value of the dose response curves of UK-14,304 for the Gi pathway in α2A-H (−PTX) versus the Gs pathway in α2A-H (+PTX) suggests that the α2A-AR couples much more efficiently to the Gi protein than to the Gs protein. This preferential coupling of the α2A-AR to Gi is in agreement with data from Eason et al. (1992, 1994) and Chabre et al. (1994). However, in HEK 293 cells transfected to transiently coexpress the porcine α2A-AR with either Gs, Gi, or Gq, Chabre et al. (1994) found that the α2A-AR couples ∼1000 times more efficiently to Gi (endogenous to HEK 293) than to either Gs (rat Gαs) or Gq (murine Gαq). The reason for the difference in the Gi/Gsselectivity of the α2A-AR coupling as suggested by the data of Chabre et al. versus our data (1000 versus 120) is not known. It is possible that coupling to endogenous G proteins is more efficient than to transfected G proteins.

It is also important to note that UK-14,304 gave no significant Gs response in α2A-L (+PTX), so the Gs response does not functionally antagonize the measured Gi response in α2A-L (−PTX). Also, contrary to what is seen in α2A-H (−PTX) at supramaximal concentrations of UK-14,304, no Gs-mediated stimulation of adenylyl cyclase is seen in α2A-L (−PTX). This is important because Gs was not inactivated by cholera toxin when the Gi responses were measured in α2A-L (−PTX).

Because the largeKi/EC50 ratio of UK-14,304 for the Gi pathway in α2A-H (−PTX) suggests a large receptor reserve, this was investigated by irreversible inhibition of the α2A-ARs by benextramine. Benextramine has been shown to irreversibly block α-ARs (Melchiorre, 1981) and has been used as such in several studies (Timmermans et al., 1985; Brasili et al., 1986; Taouis et al., 1987; Galitzky et al., 1989; Karlsson et al., 1989). Because 100 μM benextramine (20 min) shifts the dose-response curve of UK-14,304 in α2A-H (−PTX) by more than 2 logs to the right but is insufficient to reduce theEmax, it can be concluded that UK-14,304 has a large receptor reserve for the Giresponse in this cell line. In both α2A-H (+PTX) and α2A-L (−PTX), 1 μM benextramine (20 min) is sufficient to decrease theEmax, suggesting that UK-14,304 has a much smaller receptor reserve for both the Gs and Gi responses, respectively, in these two cell lines.

Conclusions.

We provide additional support for the ADTRS hypothesis in a pharmacologically well-defined system. High α2A-AR expression (higher than found in most mammalian tissue) is necessary for significant Gscoupling, which may limit the relevance of these findings for normal physiological conditions. However, the implications of this important hypothesis for drug design are highly significant. To fully understand the molecular mechanisms of ADTRS, it will be important to extend these results to purified systems with direct measurements of G protein activation.

The search for new drugs has classically been directed toward greater receptor subtype selectivity. However, ADTRS should now also be considered in the search for new drugs in that we may look for agonists that selectively activate particular effector pathways. This should also prompt reevaluation of known agonists as a means to, we hope, decrease the unwanted side effects of some drug treatments.

Acknowledgments

We thank Dr. Lee Limbird (Vanderbilt University) for providing the porcine α2A-AR cDNA. William Lim and Masakatsu Nanamori helped with some preliminary experiments related to this study.

Footnotes

  • Send reprint requests to: Richard R. Neubig, M.D., Ph.D., Department of Pharmacology, 1301 MSRB III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail:RNeubig{at}umich.edu

  • ↵1 This work was supported by National Institutes of Health Grant HL46417 and funds from Eli Lilly (Indianapolis, IN). The development of the R3 and B2 mutants was also supported by the University of Michigan Multipurpose Arthritis Center (AR20557).

  • ↵2 Present address: Division of Pharmacology, School of Pharmacy, Potchefstroom University for CHE, Potchefstroom, 2520 South Africa.

  • Abbreviations:
    ADTRS
    agonist-directed trafficking of receptor signaling
    AR
    adrenergic receptor
    α2A-H
    high α2A-adrenergic receptor expressing Chinese hamster ovary-K1 cell line
    α2A-L
    low α2A-adrenergic receptor expressing Chinese hamster ovary-K1 cell line
    α2A-B2
    B2 mutant α2A-adrenergic receptor expressing Chinese hamster ovary-K1 cell line
    α2A-R3
    R3 mutant α2A-adrenergic receptor expressing Chinese hamster ovary-K1 cell line
    5-HT
    5-hydroxytryptamine
    CHO
    Chinese hamster ovary
    ISO
    l-isoproterenol
    KA(app.)
    apparent dissociation constant of the agonist-receptor complex
    HEK
    human embryonic kidney
    TCA
    trichloroacetic acid
    DMEM
    Dulbecco's modified Eagle's medium
    PTX
    pertussis toxin
    R.e.
    relative efficacy
    IBMX
    3-isobutyl-1-methyl-xanthine
    Emax
    maximal response
    RIA
    relative intrinsic activity
    q
    fraction of receptors still functional after partial receptor alkylation
    Smax
    maximal stimulus
    • Received December 2, 1999.
    • Accepted April 19, 2000.

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Research ArticleCELLULAR AND MOLECULAR

Agonist-Directed Trafficking of Porcine α2A-Adrenergic Receptor Signaling in Chinese Hamster Ovary Cells: l-Isoproterenol Selectively Activates Gs

Christiaan B. Brink, Susan M. Wade and Richard R. Neubig
Journal of Pharmacology and Experimental Therapeutics August 1, 2000, 294 (2) 539-547;
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